This competing renewal application aims at investigating the hypothesis that exposure of human melanocytes to ultraviolet radiation (UV) and/or the physiological agonist 1-melanocyte stimulating hormone (1-MSH) or antagonist agouti signaling protein (ASIP) modulates the expression of the melanocortin 1 receptor (MC1R) gene, and regulates the activation of the receptor by affecting its desensitization, internalization and resensitization. The MC1R is a Gs protein-coupled receptor with seven transmembrane domains that is expressed on human melanocytes. Activation of this receptor by its agonists 1-MSH or ACTH stimulates cAMP formation and the synthesis of the brown-black eumelanin, which confers cutaneous photoprotection. We have shown that activation of the MC1R is pivotal for the UV-induced tanning response, and importantly, reduces the extent of UV-induced DNA damage by enhancing nucleotide excision repair and counteracting oxidative stress in human melanocytes. These effects explain why loss-of-function alleles of the MC1R are associated with increased risk for melanoma. We are proposing that MC1R expression and function are regulated at different levels in response to UV, its physiological agonists and antagonist. To investigate the above stated hypothesis, we propose the following three Specific Aims. First, we will investigate the regulation of MC1R gene expression and receptor trafficking by real time RT-PCR, Western blotting, and immunostaining. Second, we will determine the activation of the MC1R, by quantitating the number of membrane receptors/melanocyte, its agonist-induced desensitization, internalization, and resensitization. Third, we will define the roles of G protein receptor kinases (GRKs) and 2-arrestins in MC1R surface expression and sequestration. The significance of the proposed studies lies in the critical role of the MC1R and melanocortins in the UV responses of human melanocytes, and in filling the gap in the existing knowledge about regulation of this receptor in the physiologically-relevant cell, the epidermal melanocyte. Given that the MC1R is a melanoma susceptibility gene and an important determinant of the UV-induced tanning response, elucidating the regulation of MC1R expression and activation will lead to new strategies to prevent melanoma and other types of skin cancer by increasing the activity of the MC1R and optimizing the photoprotective capacity of the melanocyte, particularly in high risk individuals. PUBLIC HEALTH RELEVANCE The outcome of this grant application is expected to lead to new strategies for prevention of melanoma, the deadliest form of skin cancer, and of non-melanoma skin cancers. These strategies will be based on modulating the activity of the melanocortin 1 receptor by mechanisms that will be elucidated during the course of this grant proposal. The incidence of melanoma in the U.S. and Eastern countries continues to rise with no effective treatment for advanced disease; hence the relevance of this project for public health.